In gram-positive bacteria secreted proteins are exported across a cell membrane and a cell wall, and then are subsequently released into the external medium. On the other hand, gram-negative bacteria are surrounded by two cell (or surface) membranes; they have no cell wall. In gram-negative bacteria, most exported proteins are not released from the cell but stay in the inter-membrane periplasmic space and in the outer membrane.
Two types of components of the secretion machinery have been identified in E. coli: soluble cytoplasmic proteins and membrane associated proteins (see for review, Wickner et al., (1991) Annu. Rev. Biochem., 60:101-124). Soluble cytoplasmic proteins, including SecB and heat shock proteins, all prevent the folding of precursors of secreted proteins into a conformation incompatible with secretion. The set of membrane-associated proteins includes the peripheral membrane protein SecA, integral membrane proteins SecY, SecE, SecD, SecF and the signal peptidases Lep and Lsp (reviewed in Hayashi, S. and Wu, H. C. (1990) J. Bioenerg. Biomembr., 22:451-471; Dalbey, R. E. (1991) Mol. Microbiol., 5:2855-2860). These membrane-associated proteins are involved in binding of the precursor and in its translocation across the cytoplasmic membrane, followed by cleavage of the signal peptide and release of the protein.
Knowledge on the secretion machinery of gram-positive bacteria is more limited. The available data on B. subtilis, the genetically and physiologically well characterized model organism of the genus, suggest an overall similarity with that of E. coli, but also differences in the structure and specificity of individual components, possibly reflecting demands set by the very different composition and architecture of the respective cell envelopes.
Gram-positive bacteria such as B. subtilis, B. amyloliquefaciens, B. licheniformis have a very high capacity for secreting proteins, and indeed, many bacillar extracellular enzymes are utilized industrially. Since secreted proteins in gram-positive bacteria are so important commercially, and since the gram-positive secreted proteins traverse through a cell envelope with a very different structure from that of E. coli, the molecular mechanisms of protein secretion in gram-positive bacteria is of considerable academic and industrial importance.
In this regard a novel component of the secretion machinery of B. subtilis was recently discovered. This novel component is referred to as the PrsA protein. (See Kontinen, V. P. and Sarvas, M., (1988) J. Gen. Microbiol., 134:2333-2344; Kontinen, V. P., et al., (1991) Mol. Microbiol. 5:1273-1283). The prsA gene, which encodes the PrsA protein, was initially defined by nonlethal mutations that decreased the secretion of several exoproteins (Kontinen, V. P. and Sarvas, M., (1988) J. Gen. Microbiol., 134:2333-2344). Based on the DNA sequence of the cloned prsA gene and our subsequent work with this gene and protein, we believe prsA encodes a protein (PrsA) that acts as a chaperone, and is translocated across the cytoplasmic membrane (for the initial work, see Kontinen, V. P., et al., (1991) Mol. Microbiol. 5:1273-1283). The PrsA protein has been found to possess a limited amount of sequence homology (about 30%) with the PrtM protein of Lactococcus lactis, a protein proposed to assist the maturation of an exported serine protease (Haandrikman, A. J., et al, (1989) J. Bacteriol., 171:2789-2794; Vos, P., et al., (1989) J. Bacteriol., 171:2795-2802). A similar function has not been associated with other known proteins of the secretion machinery of bacteria, suggesting that PrsA protein is a novel type of component in the pathway of protein secretion facilitating the release and probably folding of secreted proteins after their translocation across the cytoplasmic membrane in gram-positive bacteria.
It is advantageous to produce proteins of interest in bacteria in secreted form, since exported proteins usually maintain their native conformation, in contrast to intracellular production which, in many cases, results in aggregation of the produced protein. Another advantage of producing industrially and medically important proteins in bacteria in secreted form is that secretion facilitates purification of the protein product. Additionally, unlike E. coli, gram-positive bacteria such as Bacillus sp. do not contain toxic compounds like lipopolysaccharide, making them especially appropriate hosts for production of medical and pharmaceutical proteins.
Increased yield of secreted proteins would be of great significance for improving the gram-positive bacillar strains used in the industrial production of a number of exoenzymes, such as alpha-amylases, proteases and lipases. The strategy thus far has been to overexpress the appropriate gene. There are known and readily available methods for doing this, such as increasing gene expression by using multicopy plasmids or enhancing the activity of the gene by modifying its regulatory elements, e.g., by using strong promoters, or multiple promoters. Dramatic increases of the synthesis of exoproteins have been achieved this way, up to a level at which increasing the synthesis is of no further benefit because of bottlenecks in the secretion machinery. It would be desirable to increase the capacity of secretion in parallel with increased synthesis. However, to date this has not been possible.
It is an object of the present invention to alleviate the bottleneck of the secretion mechanism in gram-positive bacteria, and to provide a method and a system whereby the levels of proteins normally secreted from gram-positive bacteria such as Bacillus can be enhanced when the expression of a given homologous or heterologous protein of interest has been elevated over the amount normally produced in unmodified or wild type organisms.
It is a further object of the present invention to describe bacterial hosts and plasmids which can be used to enhance the production of a variety of commercially important exoproteins.